Summary of work: Breast cancer incidence and mortality increase dramatically after the age of 65 and are influenced by reproductive factors, the presence of estrogen receptors (ER), and perhaps dietary factors. ER are key mediators of estrogen function and their expression can be affected by changes in methylation of the ER gene. To address the age-related mechanisms involved, we have undertaken analysis of the methylation status of exon I of the rat ER gene in spontaneous tumors of aged rats, normal contralateral mammary tissue in these tumor-bearing rats, and in mammary gland from young, tumor-free animals. We found that 48% of spontaneous tumors in aged rats exhibited low or no methylation of the ER. The contralateral normal mammary tissue from these rats was also hypomethylated. In addition, aged tumor-free rats exhibited reduced methylation in the ER gene while young and middle aged rats did not. In collaboration with scientists at Georgetown University, we have also found that induction of tumors in young rats with the chemical DMBA also leads to hypomethylation of the ER gene in the tumors as well as the normal tissue. Therefore, loss of methylation of the estrogen receptor gene is a common event in mammary tumorigenesis in the rat, increasing with age in tumors and in normal mammary gland, and during chemical induction of breast tumors in young rats. Changes in ER methylation could, therefore, serve as a predictive marker of increased breast cancer risk. Current studies are examining the role of fat intake in the incidence of tumors and in changes to ER methylation in the rat. We also have developed improved methods to grow lines of ER positive (ER+) breast tumor cells arising spontaneously in aged female rats. These cells progress to hormone-independent malignant disease in vivo and ER negative (ER-) cells can be isolated allowing the investigation of the genetic events underlying the development of these cancers. Previous studies using cell fusion of ER+ and ER- cells showed that probable loss of tumor suppressor gene(s) is important in progression. In further studies, the properties of ER+ and ER- negative cells were compared. ER+ cells were less invasive, more sensitive to programmed cell death, and expressed a different profile of proteolytic activities than ER- cells. A cDNA library of expressed genes from ER+ cells was constructed and is being used to transfect ER- cells to screen for phenotypic changes consistent with differentiated ER+ cells. This strategy will allow the isolation of genes involved in the progression to hormone-independent status. Identification of the putative suppressor gene(s) controlling the invasive phenotype may lead to therapeutic strategies to prevent breast tumor progression or to treat the malignant disease.